Study On The Sepration And Analysis Of Carbohydrate Of Natural Origin

Carbohydrate is one of the most important biological molecules in organism, which widely exist in the natural products, including monosaccharides, oligosaccharides, polysaccharides, glycosides, etc. The carbohydrates possess very complex composition, diverse chemical structure and important biological activities. The research in this thesis focused on the "separation and analysis of carbohydrate of natural origin". Based on hydrophilic interaction liquid chromatography (HILIC) method and new separation materials, pretreatment, separation and purification, structure characterization of carbohydrates were studied in detail.1. A systematic stradgy for the rapid evaluation of HILIC stationary phase was developed by using three mono-saccharides and seven di-saccharides as the model compounds. The HILIC stationary phases were silica, amide, saccharide, diol and zwitterion columns. Column temperature, column selectivity and surface charge property played important roles in the separation of carbohydrate using HILIC method. The evaluation results were valuable for the optimization of the experimental parameters such as column selectivity, mobile phase optimization and column temperature control during HILIC method development.2. A stradgy based on HILIC mode including HILIC-SPE, HILIC semi-prepration and HILIC-ELSD detection methods was applied to the separation, purification and content determination of the RFOs in water extrac of Lycopus lucidus Turcz. The quantitative analysis results proved that the root part of L. lucidus Turcz can be used as a new source of RFOs and the total content of sucrose and RFOs was 712 mg/g. This method has the characteristic of versatility, stability and validity that can be applied in the purification of oligosaccharide standards and the quality evaluation e of non-digestible oligosaccharides.3. Under the guide of "bottom-up" method in proteomics, partial acid hydrolysis-HILIC-MS/MS method was adopted for the hydrolysis, separation and structure characterization of Astragalus membranaceus oligosaccharides. Under an optimum hydrolysis condition (4 h,1.5 mol/L, and 80℃), Astragalus polysaccharides were hydrolyzed to characteristic oligosaccharide fragments. A HILIC-MS method was used for separation and structural characterization of the polysaccharides hydrolysates. The results showed that the resulting polysaccharides were mainly 1-4 linear glucan, and gluco-oligosaccharides with DP 4-11 were obtained after partial acid hydrolysis. The significance of this study was guidance for characterization of other TCM polysaccharides.4. A SPE-2D HPLC method was established for the separation of glycoside compounds especially components of low abundance in Stevia rebaudiana. The 2D HPLC method base on RPLC/HILIC showed good orthogonality for the glycosides and then it applied to purification of glycosides at preparative scale. The research results are useful to the components analysis and quality control of Stevia products.5. A novel stationary phase namely glycoside modified silica was designed and synthesized based on "thiol-ene" click chemistry strategy. The rebaudioside A (RA) was immobilized on silica surface, named "Click TE-RA". This stationary has sufficient hydrophilicity and unique selectivity for polar compounds. At the same time, under the mixed mode of HILIC/RPLC, glycosides compounds showed orthogonal retention hehavior on Click TE-RA column. On a single Click TE-RA column, selection between two modes with different separation selectivity could be achieved just by changing the composition of the mobile phase.